KANPHOS: A Database of Kinase-Associated Neural Protein Phosphorylation in the Brain

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The “KANPHOS” (Kinase-Associated Neural Phospho-Signaling) database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways.


Introduction
Protein phosphorylation and dephosphorylation are critical post-translational modifications in the regulation of cell signaling networks, especially in the nervous system, where these mechanisms contribute to brain development, functions and disorders [1][2][3][4][5]. The levels of protein phosphorylation are regulated by the combined actions of protein kinases and phosphatases. Over 500 protein kinases are encoded in the human genome, and more than 280,000 phosphorylation sites have been characterized [6]. Protein phosphorylation events in the brain have been extensively studied since the early 1970s, and it has become apparent The stable operation and updating of web-based databases requires substantial familiarity with web and network systems. To solve these problems, CMSs for the construction and management of web-based databases have become popular. The database we sought to develop needed to be able to handle a very large and continuously increasing amount of experimental data on protein kinases and phosphorylation sites. XooNIps is an easy-to-use software that can facilitate the construction of web-based databases for organizing vast amounts of information with metadata in various electronic formats and sharing it with users around the world on the internet.

Data Collection, Management and Analysis Tool
As of October 2021, approximately 10,000 records have been registered in KANPHOS. Each record is composed of a pair, a protein phosphorylation site and its modifier (i.e., the corresponding kinase or extracellular signal, such as a neurotransmitter). Of these, 6228 records were identified based on our experimental results, while 3736 records were based on publications from other groups. To facilitate analysis, KANPHOS supports three search modes: (1) search for substrates phosphorylated by a specific kinase; (2) search for kinases phosphorylating a specific protein; and (3) search for kinases and their target substrates in a specific signaling pathway. All proteins registered in KANPHOS are associated with a UniProt ID to facilitate linkage with 12 external databases (Table S1). In particular, protein associations with Gene Ontology (GO) terms and KEGG pathways are useful for exploring potential as yet unidentified functions.

Mass Spectrometry
Mass spectrometry (MS) was performed as previously described [26] with some modifications. Frozen mouse brain slices were lysed in lysis buffer (1 mM EDTA, 50 mM Tris/HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, PhosStop (Roche), and protease inhibitor cocktail (Roche), pH 7.5) and sonicated for 15 s. Supernatant was collected after centrifugation of lysate at 15,000 rpm for 10 min at 4 • C. Protein concentrations of lysates were equalized before incubation with GST-14-3-3ζ (500 pmol) immobilized on glutathione sepharose 4B beads (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) for 60 min at 4 • C. The beads were washed with lysis buffer three times, followed by wash with wash buffer (1 mM EDTA, 50 mM Tris/HCl and150 mM NaCl, pH 7.5) three times. Using guanidine solution (7 M guanidine and 50 mM Tris), bound proteins were removed from the beads, followed by reduction with 5 mM dithiothreitol for 30 min, and alkylation by10 mM iodoacetamide for 1 h at dark. The proteins were digested with Trypsin/Lys-C (Promega) and incubated overnight at 37 • C. Demineralization was performed using SPE c-tips (Nikkyo Technos, Tokyo, Japan) and phosphopeptide was enriched by TiO 2 column (Titansphere ® Phos-TiO kit, GL Sciences, Tokyo, Japan) according to the manufacturer's instructions. Finally, all the peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), which was coupled with an UltiMate3000 RSLCnano LC system (Dionex Co., Amsterdam, The Netherlands) using a nano High-performance liquid chromatography (HPLC) capillary column, C18, 3 µm, 150 mm × 75 µm (Nikkyo Technos Co., Tokyo, Japan) via a nanoelectrospray ion source. Reversed-phase chromatography was performed with solvent A (2% acetonitrile, 0.1% formic acid) and solvent B (95% acetonitrile, 0.1% formic acid) with a linear gradient (0 min, 5% B; 100 min, 40% B) at a flow rate of 300 nL/min. Prior to MS/MS analysis, a precursor ion scan was carried out using a 400-1600 mass to charge ratio (m/z) and then MS/MS analysis was performed by isolation at 0.8 Th with the quadrupole, HCD fragmentation with a normalized collision energy of 30%, and rapid scan MS analysis in the ion trap. Precursor ions with charge states of 2-6 were sampled for MS2 only. The instrument was run in a top speed mode with 3 s per cycle. Phosphoprotein identification was performed by MaxQuant software (version 1.4.1.2) (Max-Planck-Institute of Biochemistry, Computational Systems Biochemistry, Planegg, Germany) against the reference Mus musculus proteome database obtained from UniProtKB (release in November 2018) (accessed on 4 June 2021) [31]. We set carbamidomethylation of cysteine as a fixed modification, whereas methionine oxidation, Ser/Thr/Tyr phosphorylation, and N-terminal acetylation were set as variable modifications. False discovery rates (FDRs) for the peptide, protein, and site levels were set to 0.01. The ion peak intensities obtained from three independent experiments were analyzed. Gene Ontology (GO) analysis (biological process) and KEGG pathway analysis were performed using DAVID bioinformatics resources (available online: https://david.ncifcrf.gov/ (accessed on 7 November 2021)) and Reactome analysis (available online: http://www.reactome.org/, accessed on 7 November 2021).

Construction and Content of the KANPHOS Database
To construct the KANPHOS database of protein phosphorylation modification in the brain, we integrated data mainly from our own developed phosphoproteomic method (45%), from the literature (37%) and from Protoarray (18%) (Figures 1 and 2A). To obtain kinase-oriented protein phosphorylation information, we developed the KISS method for in vitro phosphoproteins [33] and the KIOSS approach for in vivo phosphorylation dependent signaling [34]. In the KISS method, an active catalytic domain of the kinase of interest is used as bait to enrich the substrates, and the interacting proteins were subsequently phosphorylated by the active catalytic domain by incubation with or without ATP ( Figure 1A). Liquid chromatography/tandem mass spectrometry (LC/MS/MS) phosphoproteomics was performed to identify the kinase-interacting proteins (candidate substrates) and their phosphorylation sites. We applied the KISS approach to a wide range of Ser/Thr and Tyr protein kinase families: Rho-kinase, Protein Kinase A (PKA), Protein Kinase B (PKB or Akt), protein kinase N (PKN), calcium/calmodulin-dependent protein kinase I (CaMK1), mitogen-activated protein kinases (MAPK), cyclin-dependent kinase 5 (CDK5), p21 (RAC1) activated kinase 7 (PAK7), Glycogen Synthase Kinase 3 Beta (GSK3β), Casein kinase 2 (CK2), Lck/Yes-related novel protein tyrosine kinase (LYN) and FYN oncogenes related to SRC, FGR, and YES (FYN) (Table 1) [25,35,36]. In the KIOSS method, cell or tissue cultures were treated with an agonist, kinase inhibitor, and/or phosphatase inhibitor, and phosphoproteins were enriched from the lysate with phospho-Ser/Thr-binding proteins, followed by LC-MS/MS analyses to identify phosphoproteins and their phosphorylation sites ( Figure 1B). We chose the 14-3-3ζ protein, tryptophan-tryptophan (WW) domain, and forkhead-associated (FHA) domain as the phospho-Ser/Thr-binding modules because these modules contain proteins that broadly regulate the function of their target proteins in a phosphorylation state-dependent manner and contribute to cellular phospho-signaling networks [37]. We performed KIOSS to analyze Rho-kinase-mediated phosphorylation in HeLa cells [27], PKA and dopamine D1 receptor-mediated phosphorylation in the mouse striatum [26]. The ratio of protein phosphorylation sites identified using the KISS method was 36%, whereas the KIOSS method identified 9% of the total entries ( Figure 2A).  In KIOSS method, mouse coronal brain slices were treated with specific kinase activator/receptor agonist or kinase inhibitor/receptor antagonist to stimulate a specific signaling pathway and then incubated with GST tag pS/T binding domain (such as 14-3-3ζ) to enrich phosphoproteins. The bound proteins were subjected to LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites. (C) In addition to our own developed phosphoproteomic method, data were also manually curated from the literature and protoarray.
Published phosphoproteomics datasets were collected from the literature by searching PubMed using keywords including 'phosphoproteomic', 'phosphoproteomics' and 'phosphoproteome'. The retrieved literature was manually reviewed and curated by our professional staff; experimentally identified candidate substrates and phosphorylation site information were collected and cross-checked with other databases, such as UniProt, Phos-phositePlus, and PhosphoELM. In addition to manual curation from the literature, data were also collected from Protoarray, which provides data generated by substrate screening for proteins phosphorylated by recombinant kinases. Each kinase was assayed at 5 nM and 50 nM in the presence of γ33P ATP to explore the phosphorylation of more than 9000 human proteins printed on the Protoarray ® microarray (Kinase Substrate Identification service, Invitrogen) [38,39]. However, these data did not contain information relating to protein phosphorylation sites. The ratio of the protein phosphorylation identified from the published literature and the Protoarray method was 37% and 18% of the total entries, respectively ( Figure 2A).

Data Accessibility and KANPHOS Workflow
To facilitate access to the KANPHOS database in a user-friendly manner, a web interface was developed with browsing and search options to facilitate the query of protein phosphorylation events. An overview of the KANPHOS workflow is shown in Figure 3. Three search options were provided on the KANPHOS homepage: 1. search for the proteins phosphorylated by selected kinases; 2. search for the kinase responsible for phos-  The distribution of phosphoproteins and their phosphorylation sites, different residue types, and species are summarized and represented in Figure 2. In total, 9964 phosphorylation sites were identified in 4141 phosphoproteins. The numbers (and percentages) of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) were 4937 (60%), 1438 (18%), and 1850 (22%), respectively ( Figure 2B); a higher ratio of pY was detected because of data obtained from FYN and LYN kinase using the KISS method. Most of the identified phosphorylation sites were phosphoserine. Although data from different species were included in KANPHOS, most of the phosphorylation events listed here are from rodents. The three species with the highest levels of phosphoproteins (phosphorylation sites) were humans (2010 (3782)), rats (1452 (4520)), and mice (680 (1662)) ( Figure 2C). Furthermore, the potential upstream kinases associated with these phosphorylation sites were annotated using kinase motif preferences ( Figure 2D). Most of the predicted phosphorylation sites belonged to the AGC family kinase (4631). The other kinase families, CAMK, CMGC, and TK, contain 1141, 1564 and 1850 phosphorylation sites, respectively. Among the predicted phosphorylation sites, 7445 (75%) were detected in vitro, 1465 (15%) were detected in vivo and approximately 1053 (10%) were detected both in vitro and in vivo ( Figure 2E).

Data Accessibility and KANPHOS Workflow
To facilitate access to the KANPHOS database in a user-friendly manner, a web interface was developed with browsing and search options to facilitate the query of protein phosphorylation events. An overview of the KANPHOS workflow is shown in Figure 3. Three search options were provided on the KANPHOS homepage: 1. search for the proteins phosphorylated by selected kinases; 2. search for the kinase responsible for phosphorylating selected substrates; and 3. search for phosphorylated proteins along with responsible kinase within pathway maps. Advanced search options are available to allow search by gene symbol/protein names, species, KEGG pathways, and Gene Ontology in a window at the left of the results list ( Figure S1). The search results can be further filtered by experimental conditions, methods, extracellular signals, in vivo/in vitro, and chemical treatments ( Figure S1). When advanced search options and/or experimental conditions are selected, only the candidate substrates that contain the string that is entered will be displayed in the search results, which enables users to quickly retrieve their data of interest. In the search results page, the results are listed, and detailed protein information can be shown by clicking the protein names. Under the selected protein, general information about the proteins, associated pathways, annotated GO terms, and phosphorylation sites can be seen on the same page. Small icons that directly link to the protein data in HUGO, rat genome database (RGD), mouse genome informatics (MGI) database, GeneCards, Allen Brain Atlas, HomoloGene, HuGENavigator, H-inv, MalaCards, and Diseases are displayed next to the general information (Table S1). Users can click the phosphorylation site information option to view the detailed information, which includes the responsible kinases, phosphorylation positions and residue types, peptide sequences, experimental methods, extracellular signals and associated drug treatment ( Figure S2).

Case Study: Identification of Adenosine-A2A Receptor Signaling and MAPK-Mediated Signaling Molecules Using KANPHOS
To examine how the KANPHOS database works, we performed KIOSS to obtain phosphoproteomic data downstream of the adenosine signaling pathway in the striatum/NAc. We used this study as a model to demonstrate the ability of the KANPHOS database to identify novel phosphorylation-dependent signaling dynamics in the brain.
rat genome database (RGD), mouse genome informatics (MGI) database, GeneCards, Allen Brain Atlas, HomoloGene, HuGENavigator, H-inv, MalaCards, and Diseases are displayed next to the general information (Table S1). Users can click the phosphorylation site information option to view the detailed information, which includes the responsible kinases, phosphorylation positions and residue types, peptide sequences, experimental methods, extracellular signals and associated drug treatment ( Figure S2). Adenosine is an important neuromodulator in the striatum/NAc that acts through adenosine A1 receptor (A1R) or adenosine A2A receptor (A2AR) [40]. Approximately 95% of striatal and accumbal neurons are medium spiny neurons (MSNs) that express either the dopamine D1 receptor (D1R-MSNs) or dopamine D2 receptor (D2R-MSNs) [41,42]. In the striatum/NAc, D2R-MSNs express A2AR, which couples to the G olf protein, whereas D1R-MSNs express A1R, which couples to the G i protein [43][44][45]. Thus, PKA activity in D2R-MSNs was positively regulated by adenosine-A2AR and negatively regulated by dopamine-D2R ( Figure 4A). To identify novel phosphoproteins downstream of adenosine-A2AR signaling, mouse striatum/NAc slices were treated with the A2AR agonist CGS21680 and the D2R agonist quinpirole ( Figure 4B). PKA activity was confirmed by monitoring the phosphorylation level of well-known PKA substrates (Rap1gap at S563, and Rasgrp2 at S116/117) by immunoblotting [46,47]. Treatment of slices with CGS21680 increased the phosphorylation level of Rap1gap at S563, and Rasgrp2 at S116/117 ( Figure 4C). The CGS21680-stimulated phosphorylation of Rap1gap at S563, and Rasgrp2 at S116/117 was blocked by pretreatment with quinpirole ( Figure 4C). These results suggest that adenosine A2AR regulates PKA activity in the striatum and NAc. Next, we performed phosphoproteomic analysis using the KIOSS method, in which affinity beads coated with the pS/T-binding domain 14-3-3ζ were used to enrich phosphorylated proteins. The bound proteins were digested using trypsin/Lys-C and loaded onto a TiO2 column to enrich for phosphopeptides, followed by LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites ( Figure 4B). Almost 80% of the phosphoproteins enhanced by A2AR agonists were inhibited by D2R agonists; thus, these phosphoproteins represent candidate substrates of adenosine signaling. These candidates were listed in Table 2. Motif analysis using the amino acid sequence logo of the identified phosphopeptides revealed that approximately 55% of the phosphorylation sites contained basic residues at the -3 and -2 positions, suggesting that the AGC group of protein kinase   Next, we performed phosphoproteomic analysis using the KIOSS method, in which affinity beads coated with the pS/T-binding domain 14-3-3ζ were used to enrich phosphorylated proteins. The bound proteins were digested using trypsin/Lys-C and loaded onto a TiO 2 column to enrich for phosphopeptides, followed by LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites ( Figure 4B). Almost 80% of the phosphoproteins enhanced by A2AR agonists were inhibited by D2R agonists; thus, these phosphoproteins represent candidate substrates of adenosine signaling. These candidates were listed in Table 2. Motif analysis using the amino acid sequence logo of the identified phosphopeptides revealed that approximately 55% of the phosphorylation sites contained basic residues at the -3 and -2 positions, suggesting that the AGC group of protein kinase such as PKA was responsible for these phosphorylation events ( Figure 4D). Proline at the +1 position was observed at about 20% of these sites, suggesting that they are phosphorylation sites for proline-oriented kinases such as MAPK1/3. Table 2. List of phosphoproteins along with phosphorylation site downstream of adenosine (A2AR) signaling.

Motif Identified Phosphoprotein and Phosphorylation Site
Ttc32 S1089 S210 T314 S316 S126 T18 We previously found that adenosine activated Rap1 through the phosphorylation of Rap1gap and Rasgrp2, thereby activating the MEK-MAPK pathway, which plays a critical role in adenosine-mediated aversive learning [48]. We next performed the KIOSS approach to explore MAPK-mediated signaling. The striatum/NAc slices were treated with a phosphatase inhibitor (okadaic acid) and/or a MEK inhibitor (U0126). Treatment of the slices with okadaic acid increased the phosphorylation of MAPK1/3 at T202/Y204, whereas pretreatment with a MEK inhibitor (U0126) reduced this okadaic acid mediated phosphorylation ( Figure 5A). Treatment with okadaic acid also increased the phosphorylation of known PKA substrate GluR1 at S845, but pretreatment with U0126 did not affect this phosphorylation, indicating that U0126 specifically inhibited MAPK1/3 activation. The cell lysates were pulled down by beads coated with 14-3-3ζ to enrich for phosphoproteins. The bound proteins were subjected to tryptic digestion and LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites. The okadaic acid treatment induced the phosphorylation of huge numbers of proteins, including MAPK substrates, and pretreatment with U0126 specifically inhibited MAPK-mediated phosphorylation. Thus, the comparison between the treatments with okadaic acid and okadaic acid with U0126 identified more than hundreds of MAPK candidate substrates ( Table 3). Comparison of MAPK candidate substrates with A2AR downstream phosphoproteins identified 22 phosphoproteins that were phosphorylated by MAPK downstream of adenosine signaling ( Figure 5C,D). There were more candidate substrates obtained by the KIOSS method than those phosphorylated by MAPK downstream of the A2AR agonist. The candidate substrates did not overlap much. This may be due to the higher sensitivity of the KIOSS method.
The identification of the related signaling pathways to the obtained phosphoproteins by using the Reactome database (available online: http://www.reactome.org/ (accessed on 7 November 2021)) revealed several potassium and Hyperpolarization-activated cyclic nucleotide gated (HCN) channels that may potentially be involved in neuronal excitability ( Figure 5B). Table 3. List of phosphoproteins identified as a MAPK candidate substrates.

Identified MAPK Candidate Substrates List
Total 157 Among the identified phosphoproteins, Arhgap21 was detected as a candidate substrate for both PKA and MAPK downstream of adenosine signaling. In the next section, we describe the use of the KANPHOS database using Arhgap21 as an example. Phosphorylated proteins can be searched by clicking the "Kinases" icon on the home page, which will open the "kinases" page. Protein kinases are listed on the left of the page, and subtype cascades can be opened by clicking the "plus" icon associated with each kinase ( Figure 6A). There are links to the IUPHAR (International union of basic and clinical pharmacology) protein kinase pages for each kinase. As we previously described, adenosine signaling activates PKA; therefore, if we choose PKA from the kinase list, the result page will be open. Using the advanced search window on the left side of the result page, we can further filter the candidate substrates list by selecting "Signal drug" (A2AR agonist) to identify phosphoproteins downstream of adenosine signaling. A list of candidate phosphoproteins will appear, listing only perfect matches to the string that was entered. From the list of candidate substrates, we chose Arhgap21 and clicked on it to obtain general information about the protein and detailed information about the phosphorylation sites ( Figure 6A). Striatal/accumbal slices were treated with okadaic acid (1 µM) for 60 min after pretreatment of MEK inhibitor U0126 (10 µM) for 30 min. OA induced the phosphorylation ERK at T202/Y204 and PKA substrate GluR1 at S845, whereas the pretreatment of MEK inhibitor reduced the phosphorylation of ERK but had no effect on PKA substrate GluR1 phosphorylation; (B) In silico analysis of the identified MAPK candidate substrates using the Reactome database (available online: http://www.reactome.org/ (accessed on 7 November 2021)) is shown; (C) Overlapping of MAPK and A2AR candidate substrates; (D) List of MAPK candidate substrates downstream of A2AR signaling. To identify the kinase responsible for phosphorylating a specific substrate, the user can click on "Substrates" icons on the home page and then enter the substrate name (Figure 6B). Here, we entered Arhgap21 and then clicked the "Search" button to generate a To identify the kinase responsible for phosphorylating a specific substrate, the user can click on "Substrates" icons on the home page and then enter the substrate name ( Figure 6B). Here, we entered Arhgap21 and then clicked the "Search" button to generate a list of available substrate of Arhgap21 in various species. We then selected only those substrate data that were obtained from mice and clicked the "Select" button to add the substrate name. This opened a list of kinases that phosphorylate Arhgap21 in mice. A similar filter option was applied in the advanced search option "Signal drug" (A2AR agonist) to identify kinases (PKA, MAPK) that phosphorylate Arhgap21 in adenosine signaling ( Figure 6B).

Pathway Analysis Using KANPHOS Revealed That HCN and Calcium Channel Proteins Are Involved in the MEK-MAPK Pathway Downstream of A2AR
Protein phosphorylation orchestrates neurotransmitter release, neuronal polarity, and neuronal signaling pathways and is involved in the pathogenesis of many neurological diseases; thus, pathway analysis using the KANPHOS database could provide a means to understand how phosphorylation events can regulate neuronal signaling events. In the previous section, we identified MAPK candidate substrates. Here, we propose a workflow for analyzing MAPK phosphoproteomics data using KANPHOS pathway analysis as a model study to identify novel phosphoproteins downstream of the MEK-MAPK pathway in A2AR (Figure 7). First, click on the "Pathways" icon on the home page, which opens the pathway viewer window. Here, we can find five main search options on the left frame: 1. neurotransmitter, 2. axon guidance, 3. neuronal polarity, 4. intracellular signaling, and 5. neuronal diseases. Additionally, each option is composed of several other search options that users can select according to their interests. As we wanted to focus on A2AR signaling, we began with the neurotransmitter section by choosing dopamine, which opened the pathway viewer image of dopamine signaling in dopamine neurons ( Figure 7A). Effector molecules are designated as green, and kinases are designated as pink. Clicking on a specific kinase opens the detailed view of its substrate list along with all phosphorylation information. MAPK was selected from the D2R-MSN signaling pathway, and 17 phosphoproteins were found to be candidate substrates of MAPK downstream of A2AR signaling ( Figure 7B). To obtain more information on the identified candidate substrates, the user can perform pathway analysis by clicking on the upper right corner of the substrate list to reveal the associated Gene Ontology terms. All the identified pathways related to the candidate substrates will appear in a tabular format according to their p-value ( Figure 7C). Here, we found several phosphoproteins that are involved in regulating hyperpolarizationactivated cyclic-nucleotide gated (HCN) channels. To further find which phosphoproteins are involved in this channel regulation process downstream of A2AR, next we go to the advance search option and from which we select Kinase (MAPK), signal drug (A2AR Agonist) and GO term (Channel) ( Figure 7D). After choosing this option, the search can be conducted and results will appear including phosphoprotein name. Here, we identified, HCN channels (Hcn2, Hcn3), and voltage-dependent R-type Calcium voltage-gated channel Alpha 1E subunit (Cacna1e) ( Figure 7E). Previously, it was reported that MAPK was involved in regulating HCN channel [49,50] and calcium channel function [51]. Thus, KANPHOS pathway analysis can help researchers to explore further associations between MAPK and HCN and calcium channel properties downstream of A2AR signaling. The bottom left corner of the pathway search window displays specific sections for neuronal diseases, e.g., Alzheimer's disease, Parkinson's disease, and schizophrenia. By clicking on a specific disease, a KANPHOS pathway search can also be initiated to identify the relationships between the neurological disease and phosphorylation-dependent signaling events ( Figure S3). In the pathway analysis of KANPHOS, a search for Alzheimer's disease pathways reveals that CDK5 and GSK3β are important kinases involved in the pathogenesis of Alzheimer's disease. Currently, 208 and 128 phosphoproteins are registered in KANPHOS as candidate substrates for CDK5 and GSK3β, respectively. For example, in these lists, CRMP2 (collapsin response mediator protein 2; also known as dihydropyrimidinase-like protein-2, DRP2 or DPYSL2) is an abundant protein in the brain that regulates axonal growth and neuronal polarity, is phosphorylated by CDK5 at S522 and GSK3β at T509, T514, and S518, and is involved in the pathogenesis of Alzheimer's disease [52][53][54]. These phosphorylations are abnormally high in the brains of Alzheimer's disease patients [55]. Thus, using KANPHOS pathway analysis, it is possible to search for kinases and their substrates involved in Alzheimer's disease, which is useful in the search for new drug targets for the treatment of Alzheimer's disease. Alzheimer's disease [52][53][54]. These phosphorylations are abnormally high in the brains of Alzheimer's disease patients [55]. Thus, using KANPHOS pathway analysis, it is possible to search for kinases and their substrates involved in Alzheimer's disease, which is useful in the search for new drug targets for the treatment of Alzheimer's disease.

Discussion
Protein phosphorylation is one of several general mechanisms that regulates the function of proteins and signal transduction in the cell, which regulates many biological processes, including cell proliferation, growth, differentiation, and metabolism. To understand the biological role of phosphorylation, it is necessary to characterize upstream signal transduction, including the kinases responsible for phosphorylation and the functional changes in downstream molecules. Phosphorylation signaling cascades vary among tissues, and completely different cellular reactions can result from the same protein phosphorylation event in different tissues due to slight differences in the cascade components. Our KANPHOS database is designed to refer not only to phosphorylation and the responsible kinases but also to related diseases, genetic polymorphisms, and knockout mice. By examining these associated data and pathway analysis of the protein phosphorylation selected in the KANPHOS search results, the signaling cascades participating in the phosphorylation of the target protein can be predicted. Compared to the other conventional databases, brain-specific, signal transduction and kinase-oriented phosphorylation data collection is unique to KANPHOS, and would provide high quality data with good usability for neuroscientists.
Our current phosphorylation data are derived mainly from the mouse striatum and from cell lines such as HeLa cells, but other tissues such as the mouse forebrain, hippocampus, liver, muscle, and cancer cell lines could be further targets of our KISS/KIOSS phosphoproteomics research. With recent improvements to the CRISPR system, kinases can be easily knocked out in various cell lines. Our in vivo protein phosphorylation screening method, KIOSS, can be used to monitor phosphorylation-dependent signaling cascades that completely lack the target kinases. However, there remain several limitations for improving in KANPHOS. The tissues and cells deposited in the database are mainly brain and neuronal cells. We need to obtain and process phosphoproteomic data from other repositories, along with empirically examining other tissues and cell lines to produce additional tissues/cell-specific phosphoproteomic profiles, to add to our database. The number of target kinases in KIOSS is limited because we need to establish effective agonist/antagonist treatment conditions for each kinase, but knockdown of each kinase by improved gene targeting methods will help us to systematically perform KIOSS analysis on kinases of interest. As a future perspective, we will continuously update KANPHOS by incorporating phosphoproteomics data based on our experimental results and links with other databases. We are preparing the data of phosphorylation-dependent signaling by several neurotransmitters in the specific brain regions, and mounting search options to narrow down the result by brain regions as the next step.
Protein kinases such as Pak3, Limk1, ERK, and RSK2 have been implicated in intellectual disability [56,57]. However, the mechanisms by which these protein kinases are involved in intellectual disability are largely unknown. Neurodevelopment is thought to be involved in the pathogenesis of psychiatric disorders such as ASD, schizophrenia, and bipolar disorder, but the causes and etiology of these diseases are unknown. For schizophrenia, the dopamine hypothesis has been proposed because D2R antagonists are widely used as therapeutic agents for schizophrenia [58]. The glutamate hypothesis has also been proposed, since long-term administration of phencyclidine, an NMDA-R antagonist, causes schizophrenia-like symptoms [59]. D2R antagonist activates the adenosine-MAPK pathway [32]. This database will be useful to understand the pathogenesis of neurodevelopmental disorders, including intellectual disability and schizophrenia, to understand the mechanism of action of D2R antagonists and to develop new therapeutic drugs.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/1 0.3390/cells11010047/s1: Figure S1: Details information and search options on result window of a phosphoprotein. Figure S2: Advance search options in KANPHOS. Figure S3: Identification of signaling network and phosphoproteins involvement in Alzheimer's disease by KANPHOS pathway analysis. Table S1: List of external databases associated with KANPHOS database.  [26], and the other data are openly available online: "https://kanphos.neuroi nf.jp" (accessed on 1 December 2021).